Vishnu Swarup

Circulating (cell-free) nucleic acids--a promising, non-invasive tool for early detection of several human diseases.

Circulating nucleic acids (CNA) are present in small amounts in the plasma of healthy individuals. However, increased levels of plasma CNA have been reported in a number of clinical disorders like cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy‐associated complications. CNA has received special attention because of its potential application as a non‐invasive, rapid and sensitive tool for molecular diagnosis and monitoring of acute pathologies and the prenatal diagnosis of fetal genetic diseases. This review throws light on the current status of blood CNA as a diagnostic marker and its potential as a powerful tool in the future.

Quantification of Circulating Plasma DNA in Friedreich's Ataxia and Spinocerebellar Ataxia Types 2 and 12

DNA triplet repeat expansion-associated ataxias, Friedreichs ataxia, and different types of spinocerebellar ataxias (SCAs) are progressive multisystem neurodegenerative disorders. The diagnosis of this wide group of inherited ataxias is essentially based on clinical findings. Cell-free circulating DNA in plasma has been considered as a powerful tool in clinical diagnosis and prognosis of several human diseases. In the present study, clinically suspected patients were assessed on the International Co-operative Ataxia Rating Scale and further confirmed by molecular analysis of DNA triplet repeats. Quantification of plasma DNA using a highly sensitive and DNA-specific PicoGreen fluorescent assay was done. We found significantly high levels (p < 0.001) of plasma DNA of 167 ± 43 ng/mL in Friedreichs ataxia patients (n = 15), 148 ± 29 ng/mL in SCA2 patients (n = 10), and 137 ± 29 ng/mL in SCA12 patients (n = 25), whereas those of healthy controls (n = 20) was only 59 ± 15 ng/mL. Therefore, we were able to distinguish between ataxia patients and healthy controls using plasma DNA. Although the precise mechanism by which plasma DNA enters into circulation is not known, significantly higher concentrations of plasma DNA appears to be due to neuronal and muscular degeneration in these patients. Identification of genes in plasma DNA, which are overexpressed or novel, can be a promising tool for the prognosis of these diseases.

Quantitative profiling and identification of differentially expressed plasma proteins in Friedreich's ataxia.

Friedreichs ataxia (FRDA) is an autosomal recessive ataxia, characterized by progressive gait ataxia, limb ataxia, dysarthria, and areflexia associated with diabetes and hypertrophic cardiomyopathy. The primary cause of FRDA is the presence of expanded DNA triplet (GAA) repeats in the first intron of the fxn gene on chromosome 9q13. The expanded DNA repeats in fxn inhibit expression of the protein frataxin, which leads to neuronal degeneration. The aim of the study was to identify differentially expressed plasma proteins in FRDA patients for their diagnostic/prognostic applications. Clinically suspected FRDA patients (n = 42) were assessed on the International Co‐Operative Ataxia Rating Scale (ICARS), and genetic confirmation was performed by analyzing (GAA) repeats via PCR. Eighteen patients were confirmed to be homozygous for FRDA, with ICARS scores of 40 ± 8. Plasma proteomics of homozygous FRDA patients and age‐ and gender‐matched healthy controls was done using two‐dimensional difference in‐gel electrophoresis and LC‐MS/MS. Quantitative proteomic analysis (fold change ≥1.5; P < 0.05) revealed 13 differentially expressed protein spots. These proteins were found to be associated with neuropathy (α1‐antitrypsin), ataxia (apolipoprotein A‐I), oxidative stress (albumin), altered lipid metabolism (apolipoprotein C‐II, C‐III), etc. Further investigations of these differentially expressed proteins can aid in identifying prognostic/diagnostic markers for FRDA.

Quantitative Profiling and Identification of Plasma Proteins of Spinocerebellar Ataxia Type 2 Patients

Background: Spinocerebellar ataxia type 2 (SCA2) is an autosomal-dominant hereditary ataxia characterized by progressive gait and limb ataxia, dysarthria, slow saccades, neuropathy and dementia. The expansion of trinucleotide CAG repeats in the coding region of the ATXN-2 gene leads to expanded polyglutamine stretch in the mutated protein which causes neuronal death. Objective: In this study, we investigated the blood plasma of SCA2 patients to find protein biomarkers. Methods: Thirty-two ataxia patients clinically suspected for SCA2 were evaluated by the International Co-operative Ataxia Rating Scale followed by genetic analysis using PCR. Plasma proteomics of SCA2 patients and age- and gender-matched healthy controls was done using 2D-difference in-gel electrophoresis, LC-MS/MS and Western blot. Results: Genetic analysis confirmed 10 of 32 suspected SCA2 patients. Proteomic data revealed nine differentially expressed proteins in SCA2. These proteins find good association with oxidative stress, calcium-dependent apoptosis, neuropathy, and cognitive impairment in SCA2 patients. Interestingly, the elevated levels of the voltage-dependent calcium channel γ-3 subunit showed a direct correlation with calcium-generated apoptosis of Purkinje cells. The cognitive deficit, a common symptom in SCA2 patients, seems to correlate with decreased levels of transthyretin and retinol-binding protein-4. Conclusions: Some of these identified proteins in SCA2 can be useful for therapeutic, diagnostic and prognostic purposes.

Single-step blood direct PCR: A robust and rapid method to diagnose triplet repeat disorders

OBJECTIVE DNA extraction prior to polymerase chain reaction (PCR) amplification in genetic diagnoses of triplet repeat disorders (TRDs) is tedious and labour-intensive and has the limitations of sample contamination with foreign DNA, including that from preceding samples. Therefore, we aimed to develop a rapid, robust, and cost-effective method for expeditious genetic investigation of TRDs from whole blood as a DNA template. METHODS Peripheral blood samples were collected from 70 clinically suspected patients of progressive ataxia. The conventional method using genomic DNA and single-step Blood-Direct PCR (BD-PCR) method with just 2μl of whole blood sample were tested to amplify triplet repeat expansion in genes related to spinocerebellar ataxia (SCA) types 1, 2, 3, 12 and Friedreichs ataxia (FRDA). Post-PCR, the allele sizes were mapped and repeat numbers were calculated using GeneMapper and macros run in Microsoft Excel programmes. RESULTS Successful amplification of target regions was achieved in all samples by both methods. The frequency of the normal and mutated allele was concordant between both methods, diagnosing 37% positive for a mutation in either of the candidate genes. The BD-PCR resulted in higher intensities of product peaks of normal and pathogenic alleles. CONCLUSIONS The nearly-accurate sizing of the normal and expanded allele was achieved in a shorter time (4-5h), without DNA extraction and any risk of cross contamination, which suggests the BD-PCR to be a reliable, inexpensive, and rapid method to confirm TRDs. This technique can be introduced in routine diagnostic procedures of other tandem repeat disorders.

Targeting PknB, an eukaryotic-like serine/threonine protein kinase of Mycobacterium tuberculosis with phytomolecules

Tuberculosis (TB), caused by Mycobacterium tuberculosis is one of the most lethal communicable disease globally. As per the WHO Global TB Report (2015), 9.6 million cases were reported in year 2014 alone. The receptor-like protein kinase, PknB is crucial for sustained mycobacterial growth. Therefore, PknB can be a potential target to develop anti-tuberculosis drugs. In present study, we performed a comparative study to investigate binding efficacies of three phytomolecules namely, Demethylcalabaxanthone, Cryptolepine hydrochloride and Ermanin. 3D structures of PknB and phytomolecules were retrieved from Protein Data Bank (PDB ID: 2FUM) and PubChem Chemical Compound Database, respectively. PknB was set to be rigid and phytochemicals were kept free to rotate. All computational simulations were carried out using Autodock 4.0 on Windows platform. In-silico study demonstrated a strong complex formation (large binding constants and low ΔG) between phytomolecules and target protein PknB of Mycobacterium tuberculosis. However, Demethylcalabaxanthone was able to bind PknB more strongly (Kb=6.8×105M-1, ΔG=-8.06kcal/mol) than Cryptolepine hydrochloride (Kb=3.06×105M-1, ΔG=-7.58kcal/mol) and Ermanin (Kb=9.8×104M-1, ΔG=-6.9kcal/mol). These in silico analysis indicate that phytomolecules are capable to target PknB protein efficiently which is vital for mycobacterial survival and therefore can be excellent alternatives to conventional anti-tuberculosis drugs.